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Venom mixtures from insects, reptiles, and mollusks have long been a source of bioactive peptides which often have alternative uses as therapeutics. While these molecules act in numerous capacities, there have been many venom components that act on the target cells through membrane disruptive mechanisms. These peptides have long been of interest as potential antimicrobial peptide platforms, but the inherent cytotoxicity of venom peptides often results in poor therapeutic potential. Despite this, efforts are ongoing to identify and characterize venom peptide which exhibit high antimicrobial activity with low cytotoxicity and modify these to further enhance the efficacy while reducing toxicity. One example is ponericin L1 from Neoponera goeldii which has been demonstrated to have good antimicrobial activity and low in vitro cytotoxicity. The L1 sequence was modified by uniformly replacing the native hydrophobic residues with either Leu, Ile, Phe, Ala, or Val. Spectroscopic and microbiological approaches were employed to investigate how the amino acid sequence changes impacted membrane interaction, secondary structure, and antimicrobial efficacy. The L1 derivatives showed varying degrees of bilayer interaction, in some cases driven by bilayer composition. Several of the variants exhibited enhanced antimicrobial activity compared to the parent strain, while others lost all activity. Interestingly, the variant containing Val lost all antimicrobial activity and ability to interact with bilayers. Taken together the results indicate that peptide secondary structure, amino acid composition, and hydrophobicity all play a role in peptide activity, although this is a delicate balance that can result in non-specific binding or complete loss of activity if specific amino acids are incorporated.

Interactions between ionic liquids and biomolecules are of great interest due to the intrinsic properties of ionic liquids and the flexibility allowed by mixing and matching cations and anions to create unique ionic liquids. A number of ionic liquid–biomolecule studies have focused on interactions with proteins, including industrially relevant enzymes. One of these, laccase from Trametes versicolor, is a naturally derived enzyme used in the breakdown of phenolic compounds in a wide variety of industries, especially useful in breakdown of lignocellulosic materials. Here, a combination of experiments and molecular dynamics (MD) simulations was used to investigate the interactions of ionic liquids with laccase. Enzyme kinetics assays indicated that ionic liquids composed of tetramethylguanidine (TMG) and either serine or threonine caused significant reduction in enzymatic activity, while kinetics was not impacted by TMG-Asp or TMG-Glu ionic liquids. Similarly, intrinsic fluorescence of laccase in the presence of TMG-Ser and TMG-Thr exhibited a shift in spectral properties consistent with structural destabilization, but again TMG-Asp and TMG-Glu had no impact. MD simulations of laccase and ABTS with/without TMG-Ser ionic liquid provided insight into the deactivation mechanism of laccase. The simulations indicated that TMG-Ser disrupts laccase’s electron transfer mechanism.

It was recently demonstrated that one can monitor ligand-induced structure fluctuations of individual thiolate-capped gold nanoclusters using resistive-pulse nanopore sensing. The magnitude of the fluctuations scales with the size of the capping ligand, and it was later shown one can observe ligand exchange in this nanopore setup. We expand on these results by exploring the different types of current fluctuations associated with peptide ligands attaching to tiopronin-capped gold nanoclusters. We show here that the fluctuations can be used to identify the attaching peptide through either the magnitude of the peptide-induced current jumps or the onset of high-frequency current fluctuations. Importantly, the peptide attachment process requires that the peptide contains a cysteine residue. This suggests that nanopore-based monitoring of peptide attachments with thiolate-capped clusters could provide a means for selective detection of cysteine-containing peptides. Finally, we demonstrate the cluster-based protocol with various peptide mixtures to show that one can identify more than one cysteine-containing peptide in a mixture.

Cationic amphiphilic polymers have been a platform to create new antimicrobial materials that act by disrupting bacterial cell membranes. While activity characterization and chemical optimization have been done in numerous studies, there remains a gap in our knowledge on the antimicrobial mechanisms of the polymers, which is needed to connect their chemical structures and biological activities. To that end, we used a single giant unilamellar vesicle (GUV) method to identify the membrane-disrupting mechanism of methacrylate random copolymers. The copolymers consist of random sequences of aminoethyl methacrylate and methyl (MMA) or butyl (BMA) methacrylate, with low molecular weights of 1600-2100 g·mol-1. GUVs consisting of an 8:2 mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), sodium salt (POPG) and those with only 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were prepared to mimic the bacterial (Escherichia coli) or mammalian membranes, respectively. The disruption of bacteria and mammalian cell membrane-mimetic lipid bilayers in GUVs reflected the antimicrobial and hemolytic activities of the copolymers, suggesting that the copolymers act by disrupting cell membranes. The copolymer with BMA formed pores in the lipid bilayer, while that with MMA caused GUVs to burst. Therefore, we propose that the mechanism is inherent to the chemical identity or properties of hydrophobic groups. The copolymer with MMA showed characteristic sigmoid curves of the time course of GUV burst. We propose a new kinetic model with a positive feedback loop in the insertion of the polymer chains in the lipid bilayer. The novel finding of alkyl-dependent membrane-disrupting mechanisms will provide a new insight into the role of hydrophobic groups in the optimization strategy for antimicrobial activity and selectivity.

Biomimetic antimicrobial polymers have been an area of great interest as the need for novel antimicrobial compounds grows due to the development of resistance. These polymers were designed and developed to mimic naturally occurring antimicrobial peptides in both physicochemical composition and mechanism of action. These antimicrobial peptide mimetic polymers have been extensively investigated using chemical, biophysical, microbiological, and computational approaches to gain a deeper understanding of the molecular interactions that drive function. These studies have helped inform SARs, mechanism of action, and general physicochemical factors that influence the activity and properties of antimicrobial polymers. However, there are still lingering questions in this field regarding 3D structural patterning, bioavailability, and applicability to alternative targets. In this review, we present a perspective on the development and characterization of several antimicrobial polymers and discuss novel applications of these molecules emerging in the field. This article is categorized under: Therapeutic Approaches and Drug DiscoveryEmerging Technologies Therapeutic Approaches and Drug DiscoveryNanomedicine for Infectious Disease.

The thermal unfolding of the copper redox protein azurin was studied in the presence of four different amino acid-based ionic liquids (ILs), all of which have tetramethylguanidium as cation. The anionic amino acid includes two with alcohol side chains, serine and threonine, and two with carboxylic acids, aspartate and glutamate. Control experiments showed that amino acids alone do not significantly change protein stability and pH changes anticipated by the amino acid nature have only minor effects on the protein. With the ILs, the protein is destabilized and the melting temperature is decreased. The two ILs with alcohol side chains strongly destabilize the protein while the two ILs with acid side chains have weaker effects. Unfolding enthalpy (ΔHunf°) and entropy (ΔSunf°) values, derived from fits of the unfolding data, show that some ILs increase ΔHunf°while others do not significantly change this value. All ILs, however, increase ΔSunf°. MD simulations of both the folded and unfolded protein conformations in the presence of the ILs provide insight into the different IL-protein interactions and how they affect the ΔHunf° values. The simulations also confirm that the ILs increase the unfolded state entropies which can explain the increased ΔSunf° values.

BACKGROUND Multiple registries have reported that >40% of high-risk atrial fibrillation patients are not taking oral anticoagulants. The purpose of our study was to determine the presence or absence of active atrial fibrillation and CHA2DS2-VASc (Congestive heart failure, Hypertension, Age ≥75 y, Diabetes mellitus, prior Stroke [or transient ischemic attack or thromboembolism], Vascular disease, Age 65-74 y, Sex category) risk factors to accurately identify high-risk atrial fibrillation (CHA2DS2-VASc ≥2) patients requiring oral anticoagulants and the magnitude of the anticoagulant treatment gap. METHODS We retrospectively adjudicated 6514 patients with atrial fibrillation documented by at least one of: billing diagnosis, electronic medical record encounter diagnosis, electronic medical record problem list, or electrocardiogram interpretation. RESULTS After review, 4555/6514 (69.9%) had active atrial fibrillation, while 1201 had no documented history of atrial fibrillation and 758 had a history of atrial fibrillation that was no longer active. After removing the 1201 patients without a confirmed atrial fibrillation diagnosis, oral anticoagulant use in high-risk patients increased to 71.1% (P < .0001 compared with 62.9% at baseline). Oral anticoagulant use increased to 79.7% when the 758 inactive atrial fibrillation patients were also eliminated from the analysis (P < .0001 compared with baseline). In the active high-risk atrial fibrillation group, there was no significant difference in the use of oral anticoagulants between men (80.7%) and women (78.8%) with a CHA2DS2-VASc ≥2, or in women with a CHA2DS2-VASc ≥3 (79.9%). CONCLUSIONS Current registries and health system health records with unadjudicated diagnoses over-report the number of high-risk atrial fibrillation patients not taking oral anticoagulants. Expert adjudication identifies a smaller treatment gap than previously described.

Background: Antimicrobial Peptides (AMPs) are an attractive alternative to traditional small molecule antibiotics as AMPs typically target the bacterial cell membrane. A Trp-rich peptide sequence derived from water buffalo (Bubalus bubalis), BuCATHL4B was previously identified as a broad-spectrum antimicrobial peptide. Objective: In this work, native Trp residues were replaced with other naturally occurring aromatic amino acids to begin to elucidate the importance of these residues on peptide activity. Methods: Minimal Inhibitory Concentration (MIC) results demonstrated activity against seven strains of bacteria. Membrane and bilayer permeabilization assays were performed to address the role of bilayer disruption in the activity of the peptides. Lipid vesicle binding and quenching experiments were also performed to gain an understanding of how the peptides interacted with lipid bilayers. Results: MIC results indicate the original, tryptophan-rich sequence, and the phenylalanine substituted sequences exhibit strong inhibition of bacterial growth. In permeabilization assays, peptides with phenylalanine substitutions have higher levels of membrane permeabilization than those substituted with tyrosine. In addition, one of the two-tyrosine substituted sequence, YWY, behaves most differently in the lowest antimicrobial activity, showing no permeabilization of bacterial membranes. Notably the antimicrobial activity is inherently species dependent, with varying levels of activity against different bacteria. Conclusion: There appears to be little correlation between membrane permeabilization and activity, indicating these peptides may have additional mechanisms of action beyond membrane disruption. The results also identify two sequences, denoted FFF and YYW, which retain antibacterial activity but have markedly reduced hemolytic activity.

Since 2007, inpatient antibiotic stewardship programs have been required for all Joint Commission–accredited hospitals in the USA. Given the frequency of ambulatory antibiotic prescribing, in June 2019, the Joint Commission released new standards for antibiotic stewardship programs in ambulatory healthcare. This report identified five elements of performance (EPs): (1) Identify an antimicrobial stewardship leader, (2) establish an annual antimicrobial stewardship goal, (3) implement evidence-based practice guidelines related to the antimicrobial stewardship goal, (4) provide clinical staff with educational resources related to the antimicrobial stewardship goal, and (5) collect, analyze, and report data related to the antimicrobial stewardship goal. We provide eight practical tips for implementing the EPs for antimicrobial stewardship: (1) Identify a collaborative leadership team, (2) partner with informatics, (3) identify national prescribing patterns, (4) perform a needs assessment based on local prescribing patterns, (5) review guidelines for diagnosis and treatment of the selected condition, (6) identify systems-level interventions to help support providers in making appropriate treatment decisions, (7) prioritize individual EPs for your institution, and (8) re-assess local data to identify areas of strength and deficiency in local practice. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11606-020-06365-1.

As we are on the cusp of the "post-antibiotic" era due to rapid spread of drug resistant bacteria, there is an urgent need for new antimicrobials that are not susceptible to bacterial resistance mechanisms. In this review, we will discuss the recent development of "polymer therapeutics" with antimicrobial activity. Learning from host-defence peptides, we propose the biomimetic design of synthetic polymers to target bacterial cell membranes, which act by compromising the membrane integrity. The discussion is extended to the future challenges and opportunities of antimicrobial polymers for clinical applications.

Antimicrobial peptides (AMPs) have been an area of great interest, due to the high selectivity of these molecules toward bacterial targets over host cells and the limited development of bacterial resistance to these molecules through evolution. The peptides are known to selectively bind to bacterial cell surfaces through electrostatic interactions, and subsequently, the peptides insert into the cell membrane and cause local disruptions of membrane integrity leading to cell death. Previous experiments showed that replacing the Leu residues in the AMP C18G with other naturally occurring hydrophobic residues resulted in side-chain-dependent activities. This work extends the investigation to non-natural hydrophobic amino acids and the effect on peptide activity. Minimal inhibitory concentration (MIC) results demonstrated that amino acid substitutions containing long flexible carbon chains maintained or increased antimicrobial activity compared to natural analogues. In solution, the peptide showed aggregation only with the most hydrophobic non-natural amino acid substitutions. Binding assays using Trp fluorescence confirm a binding preference for anionic lipids while quenching experiments demonstrated that the more hydrophobic peptides are more deeply buried in the anionic lipid bilayers compared to the zwitterionic bilayers. The most effective peptides at killing bacteria were also those which showed some level of disruption of bacterial membranes; however, one peptide sequence exhibited very strong activity and very low levels of red blood cell hemolysis, yielding a promising target for future development.

We synthesized a combinatorial library of dendrons that display a cluster of cationic charges juxtaposed with a hydrophobic alkyl chain, using the so-called "molecular umbrella" design approach. Systematically tuning the generation number and alkyl chain length enabled a detailed study of the structure-activity relationships in terms of both hydrophobic content and number of cationic charges. These discrete, unimolecular compounds display rapid and broad-spectrum bactericidal activity comparable to the activity of antibacterial peptides. Micellization was examined by pyrene emission and dynamic light scattering, which revealed that monomeric, individually solvated dendrons are present in aqueous media. The antibacterial mechanism of action is putatively driven by the membrane-disrupting nature of these cationic surfactants, which we confirmed by enzymatic assays on E. coli cells. The hemolytic activity of these dendritic macromolecules is sensitively dependent on the dendron generation and the alkyl chain length. Via structural optimization of these two key design features, we identified a leading candidate with potent broad-spectrum antibacterial activity (4-8 μg/mL) combined with outstanding hemocompatibility (up to 5000 μg/mL). This selected compound is >1000-fold more active against bacteria as compared to red blood cells, which represents one of the highest selectivity index values ever reported for a membrane-disrupting antibacterial agent. Thus, the leading candidate from this initial library screen holds great potential for future applications as a nontoxic, degradable disinfectant.

Naturally derived antimicrobial peptides have been an area of great interest because of high selectivity against bacterial targets over host cells and the limited development of bacterial resistance to these molecules throughout evolution. There are also a significant number of venom-derived peptides that exhibit antimicrobial activity in addition to activity against mammals or other organisms. Many venom peptides share the same net cationic, amphiphilic nature as host-defense peptides, making them an attractive target for development as potential antibacterial agents. The peptide ponericin L1 derived from Neoponera goeldii was used as a model to investigate the role of cationic residues and net charge on peptide activity. Using a combination of spectroscopic and microbiological approaches, the role of cationic residues and net charge on antibacterial activity, lipid bilayer interactions, and bilayer and membrane permeabilization were investigated. The L1 peptide and derivatives all showed enhanced binding to lipid vesicles containing anionic lipids, but still bound to zwitterionic vesicles. None of the derivatives were especially effective at permeabilizing lipid bilayers in model vesicles, in-tact Escherichia coli, or human red blood cells. Taken together the results indicate that the lack of facial amphiphilicity regarding charge segregation may impact the ability of the L1 peptides to effectively permeabilize bilayers despite effective binding. Additionally, increasing the net charge of the peptide by replacing the lone anionic residue with either Gln or Lys dramatically improved efficacy against several bacterial strains without increasing hemolytic activity.

Introduction: Guidelines recommend oral anticoagulants (OAC) in high risk atrial fibrillation (HRAF) patients. It has been reported that >40% of such patients are not taking an OAC. We observed that active AF often was not present in patients labeled as having AF (either because of wrong diagnosis or no AF in last 5 years). Hypothesis: We undertook a systematic electronic medical record (EMR) review to determine the presence or absence of active AF to accurately identify HRAF patients requiring an OAC. Methods: We reviewed 6514 patients with presumed AF and 2 or more outpatient visits documented by at least one of: billing diagnosis, EMR encounter diagnosis, EMR problem list, or EKG interpretation. Results: Active AF was noted in 4555/6514 patients (69.7%) after 1959 patients with no prior history of AF or inactive AF were withdrawn from further analysis. In active AF patients, 3869 had HRAF with CHA 2 DS 2 -VASc ≥2. OAC use was statistically higher (p2 DS 2 -VASc ≥3 (79.9%). In HRAF patients not taking an OAC (20.1%), expert review frequently disagreed with that recommendation suggesting that OACs can be taken in up to 92% of HRAF patients. Conclusions: 1) Current registries with un-adjudicated EMR diagnoses over-report the number of HRAF patients not taking an OAC; 2) Expert adjudication of active AF diagnosis identifies a smaller OAC treatment gap than previously described; 3) absolute contraindication or repeated refusal to take an OAC exists in less than 10% of HRAF patients; and, 4) there were no gender differences in OAC use.

Summary Combinations of ionic liquids (ILs) with antimicrobial compounds have been shown to produce synergistic activities in model liposomes. In this study, imidazolium chloride-based ILs with alkyl tail length variations are combined with commercially available, small-molecule antimicrobials to examine the potential for combinatorial and synergistic antimicrobial effects on P. aeruginosa, E. coli, S. aureus, and S. cerevisiae. The effects of these treatments in a human cell culture model indicate the cytotoxic limits of ILs paired with antimicrobials. The analysis of these ILs demonstrates that the length of the alkyl chain on the IL molecule is proportional to both antimicrobial activity and cytotoxicity. Moreover, the ILs which exhibit synergy with small-molecule antibiotics appear to be acting in a membrane permeabilizing manner. Collectively, results from these experiments demonstrate an increase in antimicrobial efficacy with specific IL + antimicrobial combinations on microbial cultures while maintaining low cytotoxicity in a mammalian cell culture model., Graphical Abstract, Highlights • 1-alkyl-3-methylimidazolium RTILs have antimicrobial properties • Alkyl chain length correlates with lethality and permeability • Combinations of RTIL + antimicrobial enhance antimicrobial effects • Combinatorial effect is not evident in human cell lines, Chemistry; Biological Sciences; Biochemistry; Microbiology; Biophysics

Naturally derived antimicrobial peptides have been an area of great interest because of high selectivity against bacterial targets over host cells and the limited development of bacterial resistance to these molecules throughout evolution. There are also a significant number of venom-derived peptides that exhibit antimicrobial activity in addition to activity against mammals or other organisms. Many venom peptides share the same net cationic, amphiphilic nature as host-defense peptides, making them an attractive target for development as potential antibacterial agents. The peptide ponericin L1 derived from Neoponera goeldii was used as a model to investigate the role of cationic residues and net charge on peptide activity. Using a combination of spectroscopic and microbiological approaches, the role of cationic residues and net charge on antibacterial activity, lipid bilayer interactions, and bilayer and membrane permeabilization were investigated. The L1 peptide and derivatives all showed enhanced binding to lipid vesicles containing anionic lipids, but still bound to zwitterionic vesicles. None of the derivatives were especially effective at permeabilizing lipid bilayers in model vesicles, in-tact Escherichia coli, or human red blood cells. Taken together the results indicate that the lack of facial amphiphilicity regarding charge segregation may impact the ability of the L1 peptides to effectively permeabilize bilayers despite effective binding. Additionally, increasing the net charge of the peptide by replacing the lone anionic residue with either Gln or Lys dramatically improved efficacy against several bacterial strains without increasing hemolytic activity.

A library of functionalized oligo(thiophene)s with precisely controlled chain length, regioregularity, sequence, and pendant moieties in the side chains was prepared by iterative convergent/divergent organometallic couplings. The cationic and facially amphiphilic structures were designed to mimic the salient physiochemical features of host defense peptides (HDPs) while concurrently exerting a photodynamic mechanism of antibacterial activity. In the dark, the oligothiophenes exert broad-spectrum and rapid bactericidal activity in the micromolar regime, which is the typical range of HDP activity. Under visible light, the antibacterial potency is enhanced by orders of magnitude, leading to potency in the nanomolar concentration range, whereas the toxicity to red blood cells (RBCs) is almost unaffected by the same visible light exposure. We attribute the potent and selective antibacterial activity to a dual mechanism of action that involves bacterial cell binding, combined with reactive oxygen species production in the bound state. Comonomer sequence and chain length dispersity play important roles in dictating the observed biological activities. The most promising candidate compound from a set of screening experiments showed antibacterial activity that is 3 orders of magnitude more potent against bacteria relative to toxicity against RBCs. Importantly, this compound did not induce resistance upon 21 subinhibitory passages, whereas the activity of ciprofloxacin was reduced 32× in the same condition. Cytotoxicity against HeLa cells in vitro is orders of magnitude weaker than antibacterial activity under visible light illumination. Thus, we have established a new class of HDP-mimetic antibacterial compounds with nanomolar activity and cell type selectivity of greater than 1300-fold. These and related compounds may be highly promising candidates in the urgent search for new topical photodynamic antibacterial formulations.

Antimicrobial peptides (AMPs) are naturally occurring components of the immune system that act against bacteria in a variety of organisms throughout the evolutionary hierarchy. There have been many studies focused on the activity of AMPs using biophysical and microbiological techniques; however, a clear and predictive mechanism toward determining if a peptide will exhibit antimicrobial activity is still elusive, in addition to the fact that the mechanism of action of AMPs has been shown to vary between peptides, targets, and experimental conditions. Nonetheless, the majority of AMPs contain hydrophobic amino acids to facilitate partitioning into bacterial membranes and a net cationic charge to promote selective binding to the anionic surfaces of bacteria over the zwitterionic host cell surfaces. This study explores the role of hydrophobic amino acids using the peptide C18G as a model system. These changes were evaluated for the effects on antimicrobial activity, peptide-lipid interactions using Trp fluorescence spectroscopy, peptide secondary structure formation, and bacterial membrane permeabilization. The results show that while secondary structure formation was not significantly impacted by the substitutions, antibacterial activity and binding to model lipid membranes were well correlated. The variants containing Leu or Phe as the sole hydrophobic groups bound bilayers with highest affinity and were most effective at inhibiting bacterial growth. Peptides with Ile exhibited intermediate behavior while those with Val or α-aminoisobutyric acid (Aib) showed poor binding and activity. The Leu, Phe, and Ile peptides demonstrated a clear preference for anionic bilayers, exhibiting significant emission spectrum shifts upon binding. Similarly, the Leu, Phe, and Ile peptides demonstrated greater ability to disrupt lipid vesicles and bacterial membranes. In total, the data indicate that hydrophobic moieties in the AMP sequence play a significant role in the binding and ability of the peptide to exhibit antibacterial activity.

Ionic liquids (ILs) have been investigated for potential antibacterial and antibiotic applications due to their ability to destabilize and permeabilize the lipid bilayers in cell membranes. Bacterial assays have shown that combining ILs with antibiotics can provide a synergistic enhancement of their antibacterial activities. We have characterized the mechanism by which the conventional ILs 1-butyl-3-methylimidazolium chloride (BMICl) and 1-butyl-3-methylimidazolium tetrafluoroborate (BMIBF4) enhance the lipid membrane permeabilization of the well-known antibiotic polymyxin B (PMB). We studied the sizes and membrane permeabilities of multilamellar and unilamellar lipid bilayer vesicles in the presence of ILs alone in aqueous solution, PMB alone, and ILs combined together with PMB. Light scattering-based experiments show that vesicle sizes dramatically increase when ILs are combined with PMB, which suggests that the materials combine to synergistically enhance lipid membrane disruption leading to vesicle aggregation. Lipid bilayer leakage experiments using tris (2,2'-bipyridyl) ruthenium (II) (Ru(bpy)32+) trapped in lipid vesicles, in which the trapped Ru(bpy)32+ fluorescence lifetime increases when it leaks out of the vesicle, show that combining BMIBF4 and PMB together permeabilize the membrane significantly more than with PMB or the IL alone. This demonstrates that ILs can assist in antibiotic permeabilization of lipid bilayers which could explain the increased antibiotic activities in the presence of ILs in solution.

Synthetic polymers have been used as a molecular platform to develop host-defense antimicrobial peptide (AMP) mimetics which are effective in killing drug-resistant bacteria. In this topical review, we will discuss the AMP-mimetic design and chemical optimization strategies as well as the biological and biophysical implications of AMP mimicry by synthetic polymers. Traditionally, synthetic polymers have been used as a chemical means to replicate the chemical functionalities and physicochemical properties of AMPs (e.g., cationic charge, hydrophobicity) to recapitulate their mode of action. However, we propose a new perception that AMP-mimetic polymers are an inherently bioactive platform as whole molecules, which mimic more than the side chain functionalities of AMPs. The tunable nature and chemical simplicity of synthetic random polymers facilitate the development of potent, cost-effective, broad-spectrum antimicrobials. The polymer-based approach offers the potential for many antimicrobial applications to be used directly in solution or attached to surfaces to fight against drug-resistant bacteria.

The continued emergence of new antibiotic resistant bacterial strains has resulted in great interest in the development of new antimicrobial treatments. Antimicrobial peptides (AMPs) are one of many potential classes of molecules to help meet this emerging need. AMPs are naturally derived sequences, which act as part of the innate immune system of organisms ranging from insects through humans. We investigated the antimicrobial peptide AP3, which is originally isolated from the winter flounder Pleuronectes americanus. This peptide is of specific interest because it does not exhibit the canonical facially amphiphilic orientation of side chains when in a helical orientation. Different analogs of AP3 were synthesized in which length, charge identity, and Trp position were varied to investigate the sequence-structure and activity relationship. We performed biophysical and microbiological characterization using fluorescence spectroscopy, CD spectroscopy, vesicle leakage assays, bacterial membrane permeabilization assays, and minimal inhibitory concentration (MIC) assays. Fluorescence spectroscopy showed that the peptides bind to lipid bilayers to similar extents, while CD spectra show the peptides adopt helical conformations. All five peptides tested in this study exhibited binding to model lipid membranes, while the truncated peptides showed no measurable antimicrobial activity. The most active peptide proved to be the parent peptide AP3 with the highest degree of leakage and bacterial membrane permeabilization. Moreover, it was found that the ability to permeabilize model and bacterial membranes correlated most closely with the ability to predict antimicrobial activity.

The location of fluorescent groups relative to the lipid bilayer can be evaluated using fluorescence quenchers embedded in the membrane and/or dissolved in aqueous solution. Quenching can be used to define the membrane topography of membrane proteins and individual membrane-embedded hydrophobic helices by combining it with the placement of fluorescent groups, including Trp, at defined sequence positions. This chapter briefly discusses various quenching methods for studies of membrane protein topography, and provides detailed protocols for dual quencher analysis (DQA), a rapid, highly sensitive, and experimentally flexible approach in which the information gained from both a membrane-embedded and aqueous quencher is combined. The advantages of the DQA method include flexibility with regard to the bilayer compositions to which it can be applied, including membranes composed of lipids of varying head group and acyl chain compositions, as well as the ability to identify mixed populations of fluorophores residing at different depths within the bilayer.

The demand for novel antimicrobial compounds is rapidly growing due to the phenomenon of antibiotic resistance in bacteria. In response, numerous alternative approaches are being taken including use of polymers, metals, combinatorial approaches, and antimicrobial peptides (AMPs). AMPs are a naturally occurring part of the immune system of all higher organisms and display remarkable broad-spectrum activity and high selectivity for bacterial cells over host cells. However, despite good activity and safety profiles, AMPs have struggled to find success in the clinic. In this review, we outline the fundamental properties of AMPs that make them effective antimicrobials and extend this into three main approaches being used to help AMPs become viable clinical options. These three approaches are the incorporation of non-natural amino acids into the AMP sequence to impart better pharmacological properties, the incorporation of AMPs in hydrogels, and the chemical modification of surfaces with AMPs for device applications. These approaches are being developed to enhance the biocompatibility, stability, and/or bioavailability of AMPs as clinical options.

In the past decade, innovative protein therapies and bio-similar industries have grown rapidly. Additionally, ionic liquids (ILs) have been an area of great interest and rapid development in industrial processes over a similar timeline. Therefore, there is a pressing need to understand the structure and function of proteins in novel environments with ILs. Understanding the short-term and long-term stability of protein molecules in IL formulations will be key to using ILs for protein technologies. Similarly, ILs have been investigated as part of therapeutic delivery systems and implicated in numerous studies in which ILs impact the activity and/or stability of protein molecules. Notably, many of the proteins used in industrial applications are involved in redox chemistry, and thus often contain metal ions or metal-associated cofactors. In this review article, we focus on the current understanding of protein structure-function relationship in the presence of ILs, specifically focusing on the effect of ILs on metal containing proteins.

The objective of our study was to determine the impact of embedding a pretest probability rule that is required during the computerized physician order-entry (CPOE) process on the appropriateness of CT angiography (CTA) of the pulmonary arteries for the diagnosis of pulmonary embolism (PE) in the emergency department (ED).Data were obtained from the electronic medical records of all adults who visited the ED from October 17, 2010, through October 17, 2012 (n = 96,507). The primary outcome was the appropriateness of pulmonary CTA. Logistic regression was used to test whether rates of appropriate use, overuse, and underuse of pulmonary CTA improved significantly after the implementation of the decision support tool when controlling for other patient characteristics.Pulmonary CTA was appropriately used in 67.2% of patients with a modified Wells score of ≥ 4, a positive d-dimer test result, or both. CTA was overused in 19.3% of patients and underused in 13.5% of patients. Each additional month after the start of the intervention was associated with a 4-percentage point increase in the odds that the modified Wells score would indicate CTA had been used appropriately (odds ratio [OR] = 1.04; 95% CI, 1.01-1.07) and significantly lowered the odds of overuse of CTA (OR = 0.93; 95% CI, 0.90-0.96) based on the modified Wells score. These changes were not associated with any significant alteration in the level of CTA utilization or the positivity rate.The addition of a mandatory field in the CPOE record was associated with a significant improvement in the appropriate ordering of pulmonary CTA but did not change the PE positive rate or CTA utilization. It seems likely that physicians gradually inflated the modified Wells scores in spite of the fact that a threshold modified Wells score was not required to perform pulmonary CTA.

Stroke is the most common complication of atrial fibrillation (AF). Guidelines recommend anticoagulant treatment in patients with CHA2DS2VASc scores of >2. Registry data suggests that almost half of patients who should be on therapeutic anticoagulation for stroke prevention in AF (SPAF) are not. Warfarin and more recently developed agents, the “novel anticoagulants” (NOACs) reduce the risk of embolic strokes. In addition, the NOACs also reduce intracranial hemorrhage (ICH) by over 50% compared to warfarin. Anticoagulation and bridging strategies involving cardioversion, catheter ablation, and invasive/surgical procedures are reviewed. The development of reversal agents for NOACs and the introduction of left atrial appendage occluding devices will evolve the use of newer strategies for preventing stroke in high risk AF patients.

We have investigated myoglobin protein denaturation using the zwitterionic detergent Empigen BB (EBB, N,N-Dimethyl-N-dodecylglycine betaine). A combination of absorbance, fluorescence, and circular dichroism spectroscopic measurements elucidated the protein denaturation and heme dissociation from myoglobin. The results indicated that Empigen BB was not able to fully denature the myoglobin structure, but apparently can induce the dissociation of the heme group from the protein. This provides a way to estimate the heme binding free energy, &Delta, Gdissociation. As ionic liquids (ILs) have been shown to perturb the myoglobin protein, we have investigated the effects of the ILs 1-butyl-3-methylimidazolium chloride (BMICl), 1-ethyl-3-methylimidazolium acetate (EMIAc), and 1-butyl-3-methylimidazolium tetrafluoroborate (BMIBF4) in aqueous solution on the &Delta, Gdissociation values. Absorbance experiments show the ILs had minimal effect on &Delta, Gdissociation values when compared to controls. Fluorescence and circular dichroism data confirm the ILs have no effect on heme dissociation, demonstrating that low concentrations ILs do not impact the heme dissociation from the protein and do not significantly denature myoglobin on their own or in combination with EBB. These results provide important data for future studies of the mechanism of IL-mediated protein stabilization/destabilization and biocompatibility studies.

Amphiphilic alpha-helices are common motifs used in numerous biological systems including membrane channels/pores and antimicrobial peptides (AMPs), and binding proteins, and a variety of synthetic biomaterials. Previously, an amphiphilic peptide with lysine-containing motifs was shown to reversibly bind the anionic porphyrin meso-Tetra(4-sulfonatophenyl)porphyrin (TPPS(4)(2−)) and promote the formation of excitonically coupled conductive J-aggregates. The work presented here focuses on the use of this amphiphilic peptide and derivatives as a potential antimicrobial agent. AMPs are naturally occurring components of the innate immune system, which selectively target and kill bacteria. Sequence derivatives were synthesized in which the position of the Trp, used as a fluorescence reporter, was changed. Additional variants were synthesized where the hydrophobic amino acids were replaced with Ala to reduce net hydrophobicity or where the cationic Lys residues were replaced with diaminopropionic acid (Dap). All peptide sequences retained the ability to bind TPPS(4)(2−) and promote the formation of J-aggregates. The peptides all exhibited a preference for binding anionic lipid vesicles compared to zwitterionic bilayers. The Trp position did not impact antimicrobial activity, but the substituted peptides exhibited markedly lower efficacy. The Dap-containing peptide was only active against E. coli and P. aeruginosa, while the Ala-substituted peptide was inactive at the concentrations tested. This trend was also evident in bacterial membrane permeabilization. The results indicate that the amphiphilic porphyrin binding peptides can also be used as antimicrobial peptides. The cationic nature is a driver in binding to lipid bilayers, but the overall hydrophobicity is important for antimicrobial activity and membrane disruption.

Antimicrobial peptides (AMPs) have been an area of great interest, due to the high selectivity of these molecules toward bacterial targets over host cells and the limited development of bacterial resistance to these molecules throughout evolution. The peptide C18G has been shown to be a selective, broad spectrum AMP with a net +8 cationic charge from seven lysine residues in the sequence. In this work, the cationic Lys residues were replaced with other natural or non-proteinogenic cationic amino acids: arginine, histidine, ornithine, or diaminopropionic acid. These changes vary in the structure of the amino acid side chain, the identity of the cationic moiety, and the pKa of the cationic group. Using a combination of spectroscopic and microbiological methods, the influence of these cationic groups on membrane binding, secondary structure, and antibacterial activity was investigated. The replacement of Lys with most other cationic residues had, at most, 2-fold effects on minimal inhibitory concentration against a variety of Gram-positive and Gram-negative bacteria. However, the peptide containing His as the cationic group showed dramatically reduced activity. All peptide variants retained the ability to bind lipid vesicles and showed clear preference for binding vesicles that contained anionic lipids. Similarly, all peptides adopted a helical conformation when bound to lipids or membrane mimetics, although the peptide containing diaminopropionic acid exhibited a decreased helicity. The peptides exhibited a wider variety of activity in the permeabilization of bacterial membranes, with peptides containing Lys, Arg, or Orn being the most broadly active. In all, the antibacterial activity of the C18G peptide is generally tolerant to changes in the structure and identity of the cationic amino acids, yielding new possibilities for design and development of AMPs that may be less susceptible to immune and bacterial recognition or in vivo degradation.

Tryptophan (Trp) is a naturally occurring amino acid, which exhibits fluorescence emission properties that are dependent on the polarity of the local environment around the Trp side chain. However, this sensitivity also complicates interpretation of fluorescence emission data. A non-natural analogue of tryptophan, β-(1-azulenyl)-L-alanine, exhibits fluorescence insensitive to local solvent polarity and does not impact the structure or characteristics of several peptides examined. In this study, we investigated the effect of replacing Trp with β-(1-azulenyl)-L-alanine in the well-known bee-venom peptide melittin. This peptide provides a model framework for investigating the impact of replacing Trp with β-(1-azulenyl)-L-alanine in a functional peptide system that undergoes significant shifts in Trp fluorescence emission upon binding to lipid bilayers. Microbiological methods including assessment of the antimicrobial activity by minimal inhibitory concentration (MIC) assays and bacterial membrane permeability assays indicated little difference between the Trp and the β-(1-azulenyl)-L-alanine-substituted versions of melittin. Circular dichroism spectroscopy showed both that peptides adopted the expected α-helical structures when bound to phospholipid bilayers and electrophysiological analysis indicated that both created membrane disruptions leading to significant conductance increases across model membranes. Both peptides exhibited a marked protection of the respective fluorophores when bound to bilayers indicating a similar membrane-bound topology. As expected, while fluorescence quenching and CD indicate the peptides are stably bound to lipid vesicles, the peptide containing β-(1-azulenyl)-L-alanine exhibited no fluorescence emission shift upon binding while the natural Trp exhibited >10 nm shift in emission spectrum barycenter. Taken together, the β-(1-azulenyl)-L-alanine can serve as a solvent insensitive alternative to Trp that does not have significant impacts on structure or function of membrane interacting peptides.

Background An issue associated with efficient bioethanol production is the fact that the desired product is toxic to the biocatalyst. Among other effects, ethanol has previously been found to influence the membrane of E. coli in a dose-dependent manner and induce changes in the lipid composition of the plasma membrane. We describe here the characterization of a collection of ethanol-tolerant strains derived from the ethanologenic Escherichia coli strain FBR5. Results Membrane permeability assays indicate that many of the strains in the collection have alterations in membrane permeability and/or responsiveness of the membrane to environmental changes such as temperature shifts or ethanol exposure. However, analysis of the strains by gas chromatography and mass spectrometry revealed no qualitative changes in the acyl chain composition of membrane lipids in response to ethanol or temperature. To determine whether these strains contain any mutations that might contribute to ethanol tolerance or changes in membrane permeability, we sequenced the entire genome of each strain. Unexpectedly, none of the strains displayed mutations in genes known to control membrane lipid synthesis, and a few strains carried no mutations at all. Interestingly, we found that four independently-isolated strains acquired an identical C → A (V244 V) silent mutation in the ferric citrate transporter gene fecA. Further, we demonstrated that either a deletion of fecA or over-expression of fecA can confer increased ethanol survival, suggesting that any misregulation of fecA expression affects the cellular response to ethanol. Conclusions The fact that no mutations were observed in several ethanol-tolerant strains suggested that epigenetic mechanisms play a role in E. coli ethanol tolerance and membrane permeability. Our data also represent the first direct phenotypic evidence that the fecA gene plays a role in ethanol tolerance. We propose that the recurring silent mutation may exert an effect on phenotype by altering RNA-mediated regulation of fecA expression. Electronic supplementary material The online version of this article (10.1186/s12866-018-1180-1) contains supplementary material, which is available to authorized users.